Fluorescent Proteins and Their Applications in Imaging Living Cells and Tissues

2010 ◽  
Vol 90 (3) ◽  
pp. 1103-1163 ◽  
Author(s):  
Dmitriy M. Chudakov ◽  
Mikhail V. Matz ◽  
Sergey Lukyanov ◽  
Konstantin A. Lukyanov
Keyword(s):  
Protein Interactions ◽  
Fluorescent Protein ◽  
Fluorescent Proteins ◽  
Super Resolution ◽  
Visible Spectrum ◽  
Living Cells ◽  
Alternative Possibilities ◽  
Structure Evolution ◽  
Marine Animals

Green fluorescent protein (GFP) from the jellyfish Aequorea victoria and its homologs from diverse marine animals are widely used as universal genetically encoded fluorescent labels. Many laboratories have focused their efforts on identification and development of fluorescent proteins with novel characteristics and enhanced properties, resulting in a powerful toolkit for visualization of structural organization and dynamic processes in living cells and organisms. The diversity of currently available fluorescent proteins covers nearly the entire visible spectrum, providing numerous alternative possibilities for multicolor labeling and studies of protein interactions. Photoactivatable fluorescent proteins enable tracking of photolabeled molecules and cells in space and time and can also be used for super-resolution imaging. Genetically encoded sensors make it possible to monitor the activity of enzymes and the concentrations of various analytes. Fast-maturing fluorescent proteins, cell clocks, and timers further expand the options for real time studies in living tissues. Here we focus on the structure, evolution, and function of GFP-like proteins and their numerous applications for in vivo imaging, with particular attention to recent techniques.

scholarly journals Visualization of Periplasmic and Cytoplasmic Proteins with a Self-Labeling Protein Tag

2016 ◽  
Vol 198 (7) ◽  
pp. 1035-1043 ◽  
Author(s):  
Na Ke ◽  
Dirk Landgraf ◽  
Johan Paulsson ◽  
Mehmet Berkmen
Keyword(s):  
Escherichia Coli ◽  
Fluorescent Protein ◽  
Fluorescent Proteins ◽  
Living Cells ◽  
Functional Protein ◽  
Content Type ◽  
Additional Tool

ABSTRACTThe use of fluorescent and luminescent proteins in visualizing proteins has become a powerful tool in understanding molecular and cellular processes within living organisms. This success has resulted in an ever-increasing demand for new and more versatile protein-labeling tools that permit light-based detection of proteins within living cells. In this report, we present data supporting the use of the self-labeling HaloTag protein as a light-emitting reporter for protein fusions within the model prokaryoteEscherichia coli. We show that functional protein fusions of the HaloTag can be detected bothin vivoandin vitrowhen expressed within the cytoplasmic or periplasmic compartments ofE. coli. The capacity to visually detect proteins localized in various prokaryotic compartments expands today's molecular biologist toolbox and paves the path to new applications.IMPORTANCEVisualizing proteins microscopically within living cells is important for understanding both the biology of cells and the role of proteins within living cells. Currently, the most common tool is green fluorescent protein (GFP). However, fluorescent proteins such as GFP have many limitations; therefore, the field of molecular biology is always in need of new tools to visualize proteins. In this paper, we demonstrate, for the first time, the use of HaloTag to visualize proteins in two different compartments within the model prokaryoteEscherichia coli. The use of HaloTag as an additional tool to visualize proteins within prokaryotes increases our capacity to ask about and understand the role of proteins within living cells.

scholarly journals Fluorescence Phenomena in Amyloid and Amyloidogenic Bionanostructures

Crystals ◽  
2020 ◽  
Vol 10 (8) ◽  
pp. 668 ◽  
Author(s):  
B. Apter ◽  
N. Lapshina ◽  
H. Barhom ◽  
B. Fainberg ◽  
A. Handelman ◽  
...  
Keyword(s):  
Amyloid Fibrils ◽  
Fluorescent Protein ◽  
Fluorescent Proteins ◽  
Protein Structures ◽  
Visible Spectrum ◽  
Biological Tissues ◽  
New Generation ◽  
Β Sheet

Nanoscale optical labeling is an advanced bioimaging tool. It is mostly based on fluorescence (FL) phenomena and enables the visualization of single biocells, bacteria, viruses, and biological tissues, providing monitoring of functional biosystems in vitro and in vivo, and the imaging-guided transportation of drug molecules. There is a variety of FL biolabels such as organic molecular dyes, genetically encoded fluorescent proteins (green fluorescent protein and homologs), semiconductor quantum dots, carbon dots, plasmonic metal gold-based nanostructures and more. In this review, a new generation of FL biolabels based on the recently found biophotonic effects of visible FL are described. This intrinsic FL phenomenon is observed in any peptide/protein materials folded into β-sheet secondary structures, irrespective of their composition, complexity, and origin. The FL effect has been observed both in natural amyloid fibrils, associated with neurodegenerative diseases (Alzheimer’s, Parkinson’s, and more), and diverse synthetic peptide/protein structures subjected to thermally induced biological refolding helix-like→β-sheet. This approach allowed us to develop a new generation of FL peptide/protein bionanodots radiating multicolor, tunable, visible FL, covering the entire visible spectrum in the range of 400–700 nm. Newly developed biocompatible nanoscale biomarkers are considered as a promising tool for emerging precise biomedicine and advanced medical nanotechnologies (high-resolution bioimaging, light diagnostics, therapy, optogenetics, and health monitoring).

scholarly journals Fusion of a fluorescent protein to the pUL25 minor capsid protein of pseudorabies virus allows live-cell capsid imaging with negligible impact on infection

2012 ◽  
Vol 93 (1) ◽  
pp. 124-129 ◽  
Author(s):  
Kevin P. Bohannon ◽  
Patricia J. Sollars ◽  
Gary E. Pickard ◽  
Gregory A. Smith
Keyword(s):  
Pseudorabies Virus ◽  
Fluorescent Protein ◽  
Fluorescent Proteins ◽  
Living Cells ◽  
Nuclear Dynamics ◽  
Infection Models ◽  
Simplex Virus

In order to resolve the location and activity of submicroscopic viruses in living cells, viral proteins are often fused to fluorescent proteins (FPs) and visualized by microscopy. In this study, we describe the fusion of FPs to three proteins of pseudorabies virus (PRV) that allowed imaging of capsids in living cells. Included in this study are the first recombinant PRV strains expressing FP–pUL25 fusions based on a design applied to herpes simplex virus type 1 by Homa and colleagues. The properties of each reporter virus were compared in both in vitro and in vivo infection models. PRV strains expressing FP–pUL25 and FP–pUL36 preserved wild-type properties better than traditional FP–pUL35 isolates in assays of plaque size and virulence in mice. The utility of these strains in studies of axon transport, nuclear dynamics and viral particle composition are documented.

scholarly journals Superfolder mTurquoise2oxoptimized for the bacterial periplasm allows high efficiencyin vivoFRET of cell division antibiotic targets

2018 ◽  
Author(s):  
Nils Y. Meiresonne ◽  
Elisa Consoli ◽  
Laureen M. Y. Mertens ◽  
Anna Chertkova ◽  
Joachim Goedhart ◽  
...  
Keyword(s):  
Cell Division ◽  
Protein Interactions ◽  
Disulfide Bonds ◽  
Fluorescent Protein ◽  
Fluorescent Proteins ◽  
Direct Interaction ◽  
Spectroscopic Properties ◽  
New Variant ◽  
Fret Pair

AbstractFluorescent proteins (FP)s are of vital importance to biomedical research. Many of the currently available fluorescent proteins do not fluoresce when expressed in non-native environments, such as the bacterial periplasm. This strongly limits the options for applications that employ multiple FPs, such as multiplex imaging or FRET. To address this issue, we have engineered a new cyan fluorescent protein based on mTurquoise2 (mTq2). The new variant is dubbed superfolder turquoise 2 ox (sfTq2ox) and is able to withstand challenging, oxidizing environments. sfTq2oxhas improved folding capabilities and can be expressed in the periplasm at higher concentrations without toxicity. This was tied to the replacement of native cysteines that may otherwise form promiscuous disulfide-bonds. The improved sfTq2oxhas the same spectroscopic properties as mTq2, i.e. high fluorescence lifetime and quantum yield. The sfTq2ox-mNeongreen FRET pair allows the detection of periplasmic protein-protein interactions with energy transfer rates exceeding 40 %. Employing the new FRET pair, we show the direct interaction of two essential periplasmic cell division proteins FtsL and FtsB and disrupt it by mutations, paving the way forin vivoantibiotic screening.

scholarly journals Fluorescent Protein-Based Indicators for Functional Super-Resolution Imaging of Biomolecular Activities in Living Cells

2019 ◽  
Vol 20 (22) ◽  
pp. 5784 ◽  
Author(s):  
Kai Lu ◽  
Cong Quang Vu ◽  
Tomoki Matsuda ◽  
Takeharu Nagai
Keyword(s):  
Image Analysis ◽  
Light Microscopy ◽  
Fluorescent Protein ◽  
Fluorescent Proteins ◽  
Super Resolution ◽  
Living Cells ◽  
Bimolecular Fluorescence Complementation ◽  
Analysis Pipeline ◽  
Nanoscale Topography ◽  
Resolution Imaging

Super-resolution light microscopy (SRM) offers a unique opportunity for diffraction-unlimited imaging of biomolecular activities in living cells. To realize such potential, genetically encoded indicators were developed recently from fluorescent proteins (FPs) that exhibit phototransformation behaviors including photoactivation, photoconversion, and photoswitching, etc. Super-resolution observations of biomolecule interactions and biochemical activities have been demonstrated by exploiting the principles of bimolecular fluorescence complementation (BiFC), points accumulation for imaging nanoscale topography (PAINT), and fluorescence fluctuation increase by contact (FLINC), etc. To improve functional nanoscopy with the technology of genetically encoded indicators, it is essential to fully decipher the photo-induced chemistry of FPs and opt for innovative indicator designs that utilize not only fluorescence intensity but also multi-parametric readouts such as phototransformation kinetics. In parallel, technical improvements to both the microscopy optics and image analysis pipeline are promising avenues to increase the sensitivity and versatility of functional SRM.

scholarly journals Substitutional landscape of a split fluorescent protein fragment using high-density peptide microarrays

PLoS ONE ◽  
2021 ◽  
Vol 16 (2) ◽  
pp. e0241461
Author(s):  
Oana N. Antonescu ◽  
Andreas Rasmussen ◽  
Nicole A. M. Damm ◽  
Ditte F. Heidemann ◽  
Roman Popov ◽  
...  
Keyword(s):  
Amino Acid ◽  
Protein Interactions ◽  
Fluorescent Protein ◽  
Fluorescent Proteins ◽  
Super Resolution ◽  
Protein Detection ◽  
High Density ◽  
Amino Acid Substitutions ◽  
Peptide Microarrays ◽  
Green Fluorescent

Split fluorescent proteins have wide applicability as biosensors for protein-protein interactions, genetically encoded tags for protein detection and localization, as well as fusion partners in super-resolution microscopy. We have here established and validated a novel platform for functional analysis of leave-one-out split fluorescent proteins (LOO-FPs) in high throughput and with rapid turnover. We have screened more than 12,000 variants of the beta-strand split fragment using high-density peptide microarrays for binding and functional complementation in Green Fluorescent Protein. We studied the effect of peptide length and the effect of different linkers to the solid support. We further mapped the effect of all possible amino acid substitutions on each position as well as in the context of some single and double amino acid substitutions. As all peptides were tested in 12 duplicates, the analysis rests on a firm statistical basis allowing for confirmation of the robustness and precision of the method. Based on experiments in solution, we conclude that under the given conditions, the signal intensity on the peptide microarray faithfully reflects the binding affinity between the split fragments. With this, we are able to identify a peptide with 9-fold higher affinity than the starting peptide.

scholarly journals Substitutional landscape of a split fluorescent protein fragment using high-density peptide microarrays

2020 ◽  
Author(s):  
Oana N. Antonescu ◽  
Andreas Rasmussen ◽  
Nicole A.M. Damm ◽  
Ditte F. Heidemann ◽  
Roman Popov ◽  
...  
Keyword(s):  
Amino Acid ◽  
Protein Interactions ◽  
Fluorescent Protein ◽  
Fluorescent Proteins ◽  
Super Resolution ◽  
Protein Detection ◽  
High Density ◽  
Amino Acid Substitutions ◽  
Peptide Microarrays ◽  
Green Fluorescent

ABSTRACTSplit fluorescent proteins have wide applicability as biosensors for protein-protein interactions, genetically encoded tags for protein detection and localization, as well as fusion partners in super-resolution microscopy. We have established and validated a novel platform for functional analysis of leave-one-out split fluorescent proteins (LOO-FPs) in high throughput and with rapid turnover. We have screened more than 12,000 strand 10 variants using high-density peptide microarrays for binding and functional complementation in Green Fluorescent Protein. We studied the effect of peptide length and the effect of different linkers to the solid support and mapped the effect of all possible amino acid substitutions on each position as well as in the context of some single and double amino acid substitutions. As all peptides were tested in 12 duplicates, the analysis rests on a firm statistical basis allowing determination of robustness and precision of the method. We showed that the microarray fluorescence correlated with the affinity in solution between the LOO-FP and peptides. A double substitution yielded a peptide with 9-fold higher affinity than the starting peptide.

Fluorescent proteins for in vivo imaging, where's the biliverdin?

2020 ◽  
Vol 48 (6) ◽  
pp. 2657-2667
Author(s):  
Felipe Montecinos-Franjola ◽  
John Y. Lin ◽  
Erik A. Rodriguez
Keyword(s):  
Hydrogen Peroxide ◽  
In Vivo Imaging ◽  
Near Infrared ◽  
Fluorescent Protein ◽  
Fluorescent Proteins ◽  
Fluorescent Imaging ◽  
Infrared Light ◽  
Red Fluorescent Protein ◽  
Color Palette

Noninvasive fluorescent imaging requires far-red and near-infrared fluorescent proteins for deeper imaging. Near-infrared light penetrates biological tissue with blood vessels due to low absorbance, scattering, and reflection of light and has a greater signal-to-noise due to less autofluorescence. Far-red and near-infrared fluorescent proteins absorb light >600 nm to expand the color palette for imaging multiple biosensors and noninvasive in vivo imaging. The ideal fluorescent proteins are bright, photobleach minimally, express well in the desired cells, do not oligomerize, and generate or incorporate exogenous fluorophores efficiently. Coral-derived red fluorescent proteins require oxygen for fluorophore formation and release two hydrogen peroxide molecules. New fluorescent proteins based on phytochrome and phycobiliproteins use biliverdin IXα as fluorophores, do not require oxygen for maturation to image anaerobic organisms and tumor core, and do not generate hydrogen peroxide. The small Ultra-Red Fluorescent Protein (smURFP) was evolved from a cyanobacterial phycobiliprotein to covalently attach biliverdin as an exogenous fluorophore. The small Ultra-Red Fluorescent Protein is biophysically as bright as the enhanced green fluorescent protein, is exceptionally photostable, used for biosensor development, and visible in living mice. Novel applications of smURFP include in vitro protein diagnostics with attomolar (10−18 M) sensitivity, encapsulation in viral particles, and fluorescent protein nanoparticles. However, the availability of biliverdin limits the fluorescence of biliverdin-attaching fluorescent proteins; hence, extra biliverdin is needed to enhance brightness. New methods for improved biliverdin bioavailability are necessary to develop improved bright far-red and near-infrared fluorescent proteins for noninvasive imaging in vivo.

Structural Evidence of Photoisomerization Pathways in Fluorescent Proteins

2019 ◽  
Author(s):  
Jeffrey Chang ◽  
Matthew Romei ◽  
Steven Boxer
Keyword(s):  
Rational Design ◽  
Fluorescent Protein ◽  
Fluorescent Proteins ◽  
Super Resolution ◽  
Unit Cell Size ◽  
Chlorine Substituent ◽  
Gfp Chromophore ◽  
The One ◽  
Green Fluorescent ◽  
Super Resolution Microscopy

<p>Double-bond photoisomerization in molecules such as the green fluorescent protein (GFP) chromophore can occur either via a volume-demanding one-bond-flip pathway or via a volume-conserving hula-twist pathway. Understanding the factors that determine the pathway of photoisomerization would inform the rational design of photoswitchable GFPs as improved tools for super-resolution microscopy. In this communication, we reveal the photoisomerization pathway of a photoswitchable GFP, rsEGFP2, by solving crystal structures of <i>cis</i> and <i>trans</i> rsEGFP2 containing a monochlorinated chromophore. The position of the chlorine substituent in the <i>trans</i> state breaks the symmetry of the phenolate ring of the chromophore and allows us to distinguish the two pathways. Surprisingly, we find that the pathway depends on the arrangement of protein monomers within the crystal lattice: in a looser packing, the one-bond-flip occurs, whereas in a tighter packing (7% smaller unit cell size), the hula-twist occurs.</p><p> </p><p> </p><p> </p><p> </p><p> </p><p> </p> <p> </p>

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